Staphylococcus aureus is a leading cause of a wide range of clinical chronic infections mainly due to the establishment of a biofilm. Biofilm, a population of bacteria within a self-produced matrix ...of extracellular polymeric substance, decreases the susceptibility to antibiotics, immune defenses and contributes to antimicrobial resistance. To date antibiotic combination has been considered a strategy to combat S. aureus infection, but this approach does not solves the main pharmacokinetic problem caused by biofilms, consisting in insufficient drug penetration within the structure. Therefore, new antimicrobial agents that could overcome this resistance need to be discovered. Fighting staphylococcal resistance and biofilm formation is an important goal of the pharmaceutical research. Some fungicide has been observed to have antibacterial effect. anyway their use as antibiotics on S.aureus has been poorly studied. The aim of this work was to investigate the effect of the fungicide itraconazole (IT) on S. aureus biofilm formation and explore by SEM the morphological alteration after treatment. A strong biofilm disaggregation and morphologically different extracellular vesicles (EV) production were observed starting from sublethal IT doses. This suggests that IT resistance phenomena on the part of S. aureus are more difficult to establish respect other antibiotics. The adjuvant properties of IT could be used to combat bacterial biofilm and/or to improve antibiotic treatment. Moreover, because the production of EV represents a secretory pathway involved in intercellular communication shared to mammalian cells, fungi, and bacteria, our study is important to increase information that can be generalized to higher organisms.
Vascular calcification is a pathological chronic condition characterized by calcium crystal deposition in the vessel wall and is a recurring event in atherosclerosis, chronic kidney disease, and ...diabetes. The lack of effective therapeutic treatments opened the research to natural products, which have shown promising potential in inhibiting the pathological process in different experimental models. This study investigated the anti-calcifying effects of Quercetin and Berberine extracts on vascular smooth muscle cells (VSMCs) treated with an inorganic phosphate solution for 7 days. Quercetin has shown the highest anti-calcifying activity, as revealed by the intracellular quantitative assay and morphological analysis. Confocal microscopy revealed downregulation of RUNX2, a key marker for calcified phenotype, which was otherwise upregulated in calcified VSMCs. To investigate the anti-inflammatory activity of Quercetin, culture media were subjected to immunometric assays to quantify the levels of IL-6 and TNF-α, and the caspase-1 activity. As expected, calcified VSMCs released a large quantity of inflammatory mediators, significantly decreasing in the presence of Quercetin. In summary, our findings suggest that Quercetin counteracted calcification by attenuating the VSMC pathological phenotypic switch and reducing the inflammatory response. In our opinion, these preliminary
findings could be the starting point for further investigations into the beneficial effects of Quercetin dietary supplementation against vascular calcification.
Connexins are a family of membrane-spanning proteins named according to their molecular weight. They are known to form membrane channels mediating cell-cell communication, which play an essential ...role in the propagation of electrical activity in the heart. Cx26 has been described in a number of tissues but not in the heart, and its mutations are frequently associated with deafness and skin diseases. The aim of this study was to assess the possible Cx26 expression in heart tissues of different mammalian species and to demonstrate its localization at level of cardiomyocytes. Samples of pig, human and rat heart and H9c2 cells were used for our research. Immunohistochemical and molecular biology techniques were employed to test the expression of Cx26. Interestingly, this connexin was found in cardiomyocytes, at level of clusters scattered over the cell cytoplasm but not at level of the intercalated discs where the other cardiac connexins are usually located. Furthermore, the expression of Cx26 in H9c2 myoblast cells increased when they were differentiated into cardiac-like phenotype. To our knowledge, the expression of Cx26 in pig, human and rat has been demonstrated for the first time in the present paper.
In developed countries, estuarine environments are often subjected to chemical pollution, whose biological impact is profitably evaluated by the use of multi-biomarker approaches on sentinel species. ...In this paper, we investigate genotoxicity and lysosomal alterations in the Mediterranean mussel (
Mytilus galloprovincialis), from the estuary of the River Cecina (Tuscany, Italy), selected as “pilot basin” within the
Water Frame Directive (2000/60 European Community). Both native and 1 month transplanted mussels were used in order to compare these two approaches in terms of sensitiveness of specific biomarker responses. Genotoxic effects were evaluated as strand breaks, by single cell gel electrophoresis (or Comet assay), and as chromosomal alterations, by the micronucleus test in gill cells. Lysosomal alterations were assessed by the neutral red retention time (in haemocytes), lipofuscin accumulation and ultrastructure (in digestive cells). Heavy metal bioaccumulation was also analysed.
Mussels from the River Cecina showed a general alteration of all the biomarkers investigated, accompanied by an elevation of tissue metal levels. However, some differences in specific responses occurred between transplanted and native mussels. Early biomarkers, such as those based on DNA and lysosomal membrane integrity, were induced at similar degree in native and transplanted mussels; while alterations resulting from cumulative events, as the increase of micronuclei frequency were much more elevated in native specimens (23.1
±
7.6) than in transplanted (9.3
±
4.7) and reference ones (5.8
±
5.2). Similarly, the comparison between lipofuscin accumulation and mean lysosomal diameter in impacted and control sites, gave significant differences exclusively with transplanted mussels. These results suggest that the parallel use of caged and native mussels in environmental biomonitoring can improve the characterization of the study area.
The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein ...(PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.
Mutations in the PTEN-induced putative kinase1 (PINK1) represent the second most frequent cause of autosomal recessive Parkinson's disease. The PINK1 protein mainly localizes to mitochondria and ...interacts with a variety of proteins, including the pro-autophagy protein beclin1 and the ubiquitin-ligase parkin. Upon stress conditions, PINK1 is known to recruit parkin at the surface of dysfunctional mitochondria and to activate the mitophagy cascade. Aim of this study was to use a simple and highly reproducible catecholamine cell model and transmission electron microscopy to characterize whether PINK1 could affect mitochondrial homeostasis, the recruitment of specific proteins at mitochondria, mitophagy and apoptosis. Samples were analyzed both in baseline conditions and following treatment with methamphetamine (METH), a neurotoxic compound which strongly activates autophagy and produces mitochondrial damage. Our data provide robust sub-cellular evidence that the modulation of PINK1 levels dramatically affects the morphology and number of mitochondria and the amount of cell death. In particular, especially upon METH exposure, PINK1 is able to increase the total number of mitochondria, concurrently recruit beclin1, parkin and ubiquitin and enhance the clearance of damaged mitochondria. In the absence of functional PINK1 and upon autophagy stress, we observe a failure of the autophagy system at large, with marked accumulation of dysfunctional mitochondria and dramatic increase of apoptotic cell death. These findings highlight the strong neuroprotective role of PINK1 as a key protein in the surveillance and regulation of mitochondrial homeostasis.
A 3 adenosine receptor activation has been previously demonstrated to result in both neuroprotective and neurodegenerative effects,
depending upon specific pathophysiological conditions. This dual ...effect may depend on receptor regulation mechanisms that
are able to change receptor availability and/or function. In the present study, we investigated desensitization, internalization,
and down-regulation of native A 3 adenosine receptors in human astrocytoma cells after exposure to the agonist 2-chloro- N 6-(3-iodobenzyl)- N -methyl-5â²-carbamoyladenosine (Cl-IBMECA). Cl-IBMECA induced a concentration-dependent inhibition of adenylyl cyclase activity
with an EC 50 value of 2.9 ± 0.1 nM. The effect was suggested to be mediated by A 3 adenosine receptor subtype by the use of selective adenosine receptor antagonists. Cell treatment with pertussis toxin abolished
Cl-IBMECA-mediated inhibition of adenylyl cyclase activity, evidencing an A 3 receptor coupling to inhibitory G protein. Short-term exposure to the agonist Cl-IBMECA (100 nM) caused rapid receptor desensitization,
within 15 min. Agonist-induced desensitization was accompanied by receptor internalization: A 3 adenosine receptor internalized with rapid kinetics, within 30 min, after cell exposure to 100 nM Cl-IBMECA. The localization
of A 3 adenosine receptors on the plasma membrane and in intracellular compartments was directly revealed by immunogold electron
microscopy. After desensitization, the removal of agonist led to the restoration of A 3 adenosine receptor functioning through receptor recycling to the cell surface within 120 min. Prolonged agonist exposure
(1â24 h) resulted in a marked down-regulation of A 3 adenosine receptors that reached 21.9 ± 2.88% of control value after 24 h. After down-regulation, the recovery of receptor
functioning was slow (24 h) and associated with the restoration of receptor levels close to control values. In conclusion,
our results demonstrated that A 3 receptors, in astrocytoma cells, are regulated after short- and long-term agonist exposure.
The response of wild chubs (
Leuciscus cephalus) to chemical pollution was assessed in a metal contaminated river (Cecina River, Italy) through a wide battery of biomarkers which included: Comet ...assay detecting DNA strand breaks; diffusion assay for apoptosis induction; micronucleus test assessing chromosomal alterations; ethoxyresorufin
O-deethylase (EROD) activity for the induction of cytochrome P 4501A; acetylcholinesterase (AChE) activity responsive to pesticide exposure; vitellogenin gene expression in males revealing estrogenic effects. Bioaccumulation of mercury, chromium and polycyclic aromatic hydrocarbons (PAHs) was also determined. Levels of mercury and PAHs were higher in tissues of chubs sampled from the most downstream station, reflecting an anthropogenic pollution of industrial origin. Otherwise, accumulation of Cr was quite similar in fish along the entire course of Cecina River confirming a natural origin due to local geochemical features. Biomarker responses revealed a significant increase of apoptotic cells, DNA stand breaks and micronucleus frequency in chubs from the more impacted sites. A slight EROD induction and AChE inhibition were only seen at the most downstream station demonstrating a limited impact due to PAHs and pesticides. On the other hand, the induction of vitellogenin gene in male chubs was measured in all the sites, suggesting a diffuse estrogenic effect. This study confirmed the utility of large batteries of biomarkers in biomonitoring studies and the suitability of wild chub as bioindicator organism for river basins.
3,4-Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative, which is neurotoxic to both serotonin (5HT) and dopamine (DA) nerve terminals. Previous reports, carried out in rodents and ...non-human primates, demonstrated neurotoxicity to monoamine axon terminals, although no study has analyzed nigral and striatal cell bodies at the sub-cellular level.
In this study, we examined intrinsic nigral and striatal cells, and PC12 cell cultures to evaluate whether, in mice, MDMA might affect nigral and striatal cell bodies.
After administering MDMA, we analyzed effects induced in vivo and in vitro using high-performance liquid chromatography (HPLC) analysis, light- and electron microscopy with immunocytochemistry, and DNA comet assay.
We found that MDMA (5 mg/kg x4, 2 h apart), besides a decrease of nigrostriatal DA innervation and 5HT loss, produces neuronal inclusions within nigral and intrinsic striatal neurons consisting of multi-layer ubiquitin-positive whorls extending to the nucleus of the cell. These fine morphological changes are associated with clustering of heat shock protein (HSP)-70 in the nucleus, very close to chromatin filaments. In the same experimental conditions, we could detect oxidation of DNA bases followed by DNA damage. The nature of inclusions was further investigated using PC12 cell cultures.
The present findings lead to re-consideration of the neurotoxic consequences of MDMA administration. In fact, occurrence of ubiquitin-positive neuronal inclusions and DNA damage both in nigral and striatal cells sheds new light into the fine alterations induced by MDMA, also suggesting the involvement of nuclear and cytoplasmic components of the ubiquitin-proteasome pathway in MDMA toxicity.
Abstract Methamphetamine produces locomotor activation and typical stereotyped motor patterns, which are commonly related with increased catecholamine activity within the basal ganglia, including the ...dorsal and ventral striatum. Since the cerebellum is critical for movement control, and for learning of motor patterns, we hypothesized that cerebellar catecholamines might be a target of methamphetamine. To test this experimental hypothesis we injected methamphetamine into C57 Black mice at the doses of 5 mg/kg two or three times, 2 h apart. This dosing regimen is known to be toxic for striatal dopamine terminals. However, we found that in the cerebellum, methamphetamine increased the expression of the primary transcript of the tyrosine hydroxylase (TH) gene, followed by an increased expression of the TH protein. Increased TH was localized within Purkinje cells, where methamphetamine increased the number of TH-immunogold particles, and produced a change in the distribution of the enzyme by increasing the cytoplasmic percentage. Increased TH expression was accompanied by a slight increase in noradrenaline content. This effect was highly site-specific for the cortex of posterior vermal lobules, while only slight effects were detectable in the hemispheres. The present data indicate that the cerebellum does represent a target of methamphetamine, which produces specific and fine alterations of the catecholamine system involving synthesis, amount, and compartmentalization of TH as well as increased noradrenaline levels. This may be relevant for motor alterations induced by methamphetamine. In line with this, inherited cerebellar movement disorders in various animal species including humans are associated with increased TH immunoreactivity within intrinsic neurons of the same lobules of the cerebellar cortex.
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